1. Field of the Invention
The invention relates to a method for characterizing the cellular components of a biological fluid and for quantifying the populations and subpopulations of cells in the biological fluid, and a device which can be used to carry out the method.
Within the scope of the invention the phrase “biological fluid” refers to any natural fluid or biological preparation containing cellular components or capable of containing such components, such as blood, bone marrow, cerebrospinal fluid, pleural fluid, and the phrase “cellular component” also denotes a cell in its most conventional sense, such as red and white blood corpuscles, as well as any other cell type present in a biological fluid, such as platelets. Equally, the word “population” refers to a set of cells of the same category, for example, basophiles, lymphocytes, etc., and the term “subpopulation” denotes a subassembly of specific cells of the same category, such as B, T4, T8 lymphocytes, etc., of the lymphocyte population, but also immature or degenerate forms.
2. Description of Related Art
Determining and counting the entire cell population in various biological fluids such as blood are of major importance in terms of clinical diagnosis. The traditional methods of carrying out cytological analyses of a biological fluid make use of various conventional apparatus such as hematology machines based on flow cytometry, which are intended for differentiation and accurate automatic counting of all the cells present in a sample of biological fluid, which can then be classified into populations. These apparatus normally include measuring equipment, typically electrical, and/or optical, compatible with the possible presence of various reagents and dyes, leading to analysis results as defined above. Thus, for a blood sample, these automatic machines can draw up a hemogram which provides the classic parameters of a blood count, of the leukocyte formula and of platelet count. Using additional apparatus which are separate from one another it is possible to obtain additional results on the identification of reticulocytes, erythroblasts, immature cells, etc. in order to finalize the data of the hemogram. By way of example, reference may be made to patent application FR0102489 filed by the present applicant, which relates to a method and a reagent for the identification and counting of biological cells by multi-parametric methods of measurement making use of flow cytometry.
Thus it is possible to deduce information for each of the categories of biological cells, resulting in their classification.
In the great majority of cases the accurate automatic counting of all the cellular components and the differentiation of each of the populations of cellular components are sufficient to give the practitioner information as to the presence of an imbalance or a cytological disorder. However, when analyzing a biological fluid using a conventional automatic cytological analyzer, certain abnormal cell count results, compared with those which are normally expected, may justify further investigation in order to refine one or more of these results. Furthermore, such analysis does not accurately quantify the different populations of cells within a single population or family, which is a major drawback, particularly for detecting certain pathological conditions in patients or for monitoring their development.
Complementary analysis thus proves essential in order to achieve more accurate results on the count of a selected subpopulation of cells, e.g. leukocytes, in a blood sample. The appearance of certain pathological conditions (immune reactions or leukemia) may be correlated with an abnormal level of a leukocyte-type cell population.
Complementary analysis of this kind, independent of that carried out using a conventional machine, may consist in taking a manual smear followed by observation under the microscope. Identification and counting of cellular components are, in this case, currently used in addition to or as a replacement for those obtained using the automated equipment.
They may also consist of specific cell labeling using selected antibodies, possibly including fluorochromes, for discriminating a selected labeled cell population, or using any other labeling method known from the prior art. For example, mention may be made of patent application EP0552707, which discloses labeling using anti-CD45 and anti-CD71 monoclonal antibodies, for example, in order to differentiate all the leukocyte cells in flow cytrometry. Patent Application WO00/16103 describes the quantification of eosinophilic and basophilic cells by discrimination thereof carried out using antibodies or an antibody kit and detecting them by fluorometry. Equally, the labeling of a lymphocyte population with anti-CD19, anti-CD4 and anti-CD8 antibodies makes it possible to separate the subpopulations of B lymphocytes, T helper lymphocytes and T suppressor lymphocytes, respectively.
Although these analyses can be used to monitor and/or correct the results obtained with a conventional automatic apparatus, they often introduce a degree of inaccuracy and allow only a statistical correlation with the results obtained using the automatic apparatus. The use of higher-performance methods such as flow cytometry produces substantially the same results, but a direct correlation with the results obtained using the automatic apparatus is not possible because two separate analyses are carried out, thus reducing the accuracy of the results as a whole.
In fact, when an analysis of the cellular components of an aliquot of biological fluid reveals an anomaly in the precise count of cellular components, the user goes on to perform a second independent analysis on a different aliquot of the same biological fluid, using two independent analysis systems, thus carrying out measurements on separate bases, i.e. each of the measurements supplies different results, and therefore mathematical correlation of the results obtained by each of the measurements is needed in order to obtain, eventually, results which contain errors. This leads to an interpretation of the results which takes on a subjective nature as it does not allow reliable identification of the real cause of the abnormal results, which may be due either to the sample itself or to the equipment and may also prove unacceptable for the precise diagnosis of a pathological condition, in some cases.